The long term goal of this proposal is to understand how circadian clock networks are constructed in eukaryotic cells. Circadian clocks are known to regulate many essential cellular processes widely distributed across biological systems, including humans. In higher plants, the clock network regulates diverse processes ranging from photosynthesis to cell elongation to the control of flowering time. We have chosen Arabidopsis as a model organism and have identified several clock genes from genetic screens. The reciprocal regulation amongst these clock components defined an initial molecular feedback loop which forms the basis for elaborated models of multiple loop clock networks in higher plants. We have made substantial progress on all of the previous specific aims, including the identification and characterization of several new clock genes. The experiments in this proposal aim to build on the current clock models by continuing to identify clock factors involved in regulating the transcription of known key clock components such as CCA1 and TOC1. To this end, we have created a unique functional genomics resource consisting of a library of more than 200 cycling transcription factors that can be used to detect DNA binding as well as protein-protein interactions on a spectrum of targets. This has led to the discovery of a novel transcription factor TCP21 that binds to the CCA1 promoter. This proposal aims to characterize TCP21 and identify additional transcriptional regulators within the core network. We have also identified a novel clock transcription factor LUX that will be characterized in terms of its DNA target genes and interaction partners. We propose to extensively characterize the time dependent protein-protein interactions and post- translational mechanisms that add critical additional layers of control to the transcriptional components of the clock. Finally, we plan to use a systems biology approach to characterize the logic underlying the output networks of the clock. We wish to explore the similarities in system architecture between plant and animal clock networks. Given the ubiquity of circadian-regulated physiology, characterization of circadian systems in model organisms will impact our understanding of the pacemaker mechanisms and malfunctions associated with many known features of human well-being.